Federico I. Rosell

Side-On Copper-Nitrosyl Coordination by Nitrite Reductase

A copper-nitrosyl intermediate forms during the catalytic cycle of nitrite reductase, the enzyme that mediates the committed step in bacterial denitrification. The crystal structure of a type 2 copper-nitrosyl complex of nitrite reductase reveals an unprecedented side-on binding mode in which the nitrogen and oxygen atoms are nearly equidistant from the copper cofactor. Comparison of this structure with a refined nitrite-bound crystal structure explains how coordination can change between copper-oxygen and copper-nitrogen during catalysis. The side-on copper-nitrosyl in nitrite reductase expands the possibilities for nitric oxide interactions in copper proteins such as superoxide dismutase and prions.

Site-directed mutagenesis improves the biocatalytic activity of iso-1-cytochrome c in polycyclic hydrocarbon oxidation

Abstract iso-1-Cytochrome c from Saccharomyces cerevisiae is able to oxidize polycyclic aromatic hydrocarbons (PAH) in the presence of hydrogen peroxide. Anthracene and pyrene are oxidized by yeast cytochrome c to form anthraquinone and 1,8-pyrenedione, respectively. Iso-1-cytochrome c from S. cerevisiae was modified by site-directed mutagenesis of Phe82 and Cys102. The Phe82 substitution significantly altered the kinetic behavior of the protein; Cys102 modification affected neither the kinetic nor the stability constant. The Gly82;Thr102 variant was 10 times more active and showed a catalytic efficiency 10-fold greater than the wild-type iso-1-cytochrome c. However, Phe82 variants showed lower stability against inactivation by hydrogen peroxide than the wild-type protein. These site-directed mutations did not significantly alter the stability and activity of the hemoprotein in increasing concentrations of tetrahydrofuran.

Converting cytochrome C into a peroxidase-like metalloenzyme by molecular design.

Hemoproteins, which have a heme prosthetic group, are excellent natural models for artificial design of desired metalloproteins. Various hemoproteins can perform a range of functions by means of combining heme with a different protein scaffold, especially the coordination environment of the heme, which includes the axial ligand, coordination number, and coordination sphere. Consequently, the de novo design of a heme pocket could be a useful strategy for constructing new hemoproteins with desired or novel properties and functions. Although cytochrome c (Cyt c), an electron transfer protein, has no peroxidase activity in living systems, previous studies have shown that it can catalyze a variety of oxidation reactions in the presence of H2O2. [3] Furthermore, characterized by the covalent attachment of a heme to the polypeptide, Cyt c is a very stable protein and has several advantages for use as a peroxidase mimic. 4] In addition, it was recently discovered that the release of Cyt c from mitochondria in the initial stages of apoptosis is related to the cardiolipin oxygenase activity of the cytochrome. This provides a compelling basis for understanding the structural features of the protein that dictates the chemical reactivity of the heme. However, the intrinsic peroxidase activity of Cyt c is suppressed by the protein matrix when compared to typical peroxidases, such as horseradish peroxidase (HRP) and cytochrome c peroxidase (CcP), which have a penta-coordination heme iron and a distal histidine in the heme pocket. Therefore, replacing the sixth axial ligand (Met80) with a non-coordination amino acid and introducing a distal histidine in the heme pocket could convert Cyt c into a peroxidase-like metalloenzyme. To achieve the above-mentioned purpose, we investigated the peroxidase activity of yeast iso-1-cytochrome c (PDB ID: 2YCC) variants in which selected substitutions at the active site had been introduced in an initial effort to mimic some of the structural features of classic peroxidases, such as HRP and CcP. Molecular modeling suggests that replacement of Tyr67 with histidine should place the Ne of His67 at ~5.2 B from the heme iron. This distance was obtained from an energy-minimized simulation of a molecular model based on the PDB file 2YCC, which was created with VMD and NAMD. This distance approximates to that between the Ne of the distal histidine and the heme iron in HRP (PDB ID: 1H5A) and CcP (PDB ID: 2CYP; 5.84 and 5.55 B, respectively). This result, combined with the lack of a coordinated axial ligand in either HRP or CcP, led us to construct and evaluate the peroxidase activity of Cyt c Met80Val, Tyr67His, and Tyr67His/Met80Val variants in which either the sixth axial ligand (Met80) was eliminated and/ or a distal histidine at position 67 was introduced in the heme pocket (Figure 1).

Electron transfer in the Rhodobacter sphaeroides reaction center assembled with zinc bacteriochlorophyll

The cofactor composition and electron-transfer kinetics of the reaction center (RC) from a magnesium chelatase (bchD) mutant of Rhodobacter sphaeroides were characterized. In this RC, the special pair (P) and accessory (B) bacteriochlorophyll (BChl) -binding sites contain Zn-BChl rather than BChl a. Spectroscopic measurements reveal that Zn-BChl also occupies the H sites that are normally occupied by bacteriopheophytin in wild type, and at least 1 of these Zn-BChl molecules is involved in electron transfer in intact Zn-RCs with an efficiency of >95% of the wild-type RC. The absorption spectrum of this Zn-containing RC in the near-infrared region associated with P and B is shifted from 865 to 855 nm and from 802 to 794 nm respectively, compared with wild type. The bands of P and B in the visible region are centered at 600 nm, similar to those of wild type, whereas the H-cofactors have a band at 560 nm, which is a spectral signature of monomeric Zn-BChl in organic solvent. The Zn-BChl H-cofactor spectral differences compared with the P and B positions in the visible region are proposed to be due to a difference in the 5th ligand coordinating the Zn. We suggest that this coordination is a key feature of protein–cofactor interactions, which significantly contributes to the redox midpoint potential of H and the formation of the charge-separated state, and provides a unifying explanation for the properties of the primary acceptor in photosystems I (PS1) and II (PS2).

NADH Oxidase Activity of Indoleamine 2,3-Dioxygenase

The heme enzyme indoleamine 2,3-dioxygenase (IDO) was found to oxidize NADH under aerobic conditions in the absence of other enzymes or reactants. This reaction led to the formation of the dioxygen adduct of IDO and supported the oxidation of Trp to N-formylkynurenine. Formation of the dioxygen adduct and oxidation of Trp were accelerated by the addition of small amounts of hydrogen peroxide, and both processes were inhibited in the presence of either superoxide dismutase or catalase. Anaerobic reaction of IDO with NADH proceeded only in the presence of a mediator (e.g. methylene blue) and resulted in formation of the ferrous form of the enzyme. We propose that trace amounts of peroxide previously proposed to occur in NADH solutions as well as solid NADH activate IDO and lead to aerobic formation of superoxide and the reactive dioxygen adduct of the enzyme.

Role of Rhodobacter sphaeroides Photosynthetic Reaction Center Residue M214 in the Composition, Absorbance Properties, and Conformations of HA and BA Cofactors

In the native reaction center (RC) of Rhodobacter sphaeroides, the side chain of (M)L214 projects orthogonally toward the plane and into the center of the A branch bacteriopheophytin (BPhe) macrocycle. The possibility that this side chain is responsible for the dechelation of the central Mg(2+) of bacteriochlorophyll (BChl) was investigated by replacement of (M)214 with residues possessing small, nonpolar side chains that can neither coordinate nor block access to the central metal ion. The (M)L214 side chain was also replaced with Cys, Gln, and Asn to evaluate further the requirements for assembly of the RC with BChl in the HA pocket. Photoheterotrophic growth studies showed no difference in growth rates of the (M)214 nonpolar mutants at a low light intensity, but the growth of the amide-containing mutants was impaired. The absorbance spectra of purified RCs indicated that although absorbance changes are associated with the nonpolar mutations, the nonpolar mutant RC pigment compositions are the same as in the wild-type protein. Crystal structures of the (M)L214G, (M)L214A, and (M)L214N mutants were determined (determined to 2.2-2.85 Å resolution), confirming the presence of BPhe in the HA pocket and revealing alternative conformations of the phytyl tail of the accessory BChl in the BA site of these nonpolar mutants. Our results demonstrate that (i) BChl is converted to BPhe in a manner independent of the aliphatic side chain length of nonpolar residues replacing (M)214, (ii) BChl replaces BPhe if residue (M)214 has an amide-bearing side chain, (iii) (M)214 side chains containing sulfur are not sufficient to bind BChl in the HA pocket, and (iv) the (M)214 side chain influences the conformation of the phytyl tail of the BA BChl.

Investigation of the role of a surface patch in the self-association of Chromatium vinosum high potential iron-sulfur protein

The role of a flattened, relatively hydrophobic surface patch in the self-association of Chromatium vinosum HiPIP was assessed by substituting phenylalanine 48 with lysine. The reduction potential of the F48K variant was 26 mV higher than that of the wild-type (WT) recombinant (rc) HiPIP, consistent with the introduction of a positive charge close to the cluster. Nuclear magnetic resonance spectroscopy (NMR) revealed that the electronic structure of the oxidized cluster in these two proteins is very similar at 295 K. In contrast, the electron transfer self-exchange rate constant of F48K was at least 15-fold lower than that of the WT rcHiPIP, indicating that the introduction of a positive charge at position 48 diminishes self-association of the HiPIP in solution. Moreover, the substitution at position 48 abolished the fine structure in the g(z) region of the electron paramagnetic resonance (EPR) spectrum of oxidized C. vinosum rcHiPIP recorded in the presence of 1 M sodium chloride. These results support the hypothesis that the flattened, relatively hydrophobic patch mediates interaction between two molecules of HiPIP and that freezing-induced dimerization of the HiPIP mediated by this patch is responsible for the unusual fine structure observed in the EPR spectrum of the oxidized C. vinosum HiPIP.

Alteration of blood clotting and lung damage by protamine are avoided using the heparin and polyphosphate inhibitor UHRA

Anticoagulant therapy-associated bleeding and pathological thrombosis pose serious risks to hospitalized patients. Both complications could be mitigated by developing new therapeutics that safely neutralize anticoagulant activity and inhibit activators of the intrinsic blood clotting pathway, such as polyphosphate (polyP) and extracellular nucleic acids. The latter strategy could reduce the use of anticoagulants, potentially decreasing bleeding events. However, previously described cationic inhibitors of polyP and extracellular nucleic acids exhibit both nonspecific binding and adverse effects on blood clotting that limit their use. Indeed, the polycation used to counteract heparin-associated bleeding in surgical settings, protamine, exhibits adverse effects. To address these clinical shortcomings, we developed a synthetic polycation, Universal Heparin Reversal Agent (UHRA), which is nontoxic and can neutralize the anticoagulant activity of heparins and the prothrombotic activity of polyP. Sharply contrasting protamine, we show that UHRA does not interact with fibrinogen, affect fibrin polymerization during clot formation, or abrogate plasma clotting. Using scanning electron microscopy, confocal microscopy, and clot lysis assays, we confirm that UHRA does not incorporate into clots, and that clots are stable with normal fibrin morphology. Conversely, protamine binds to the fibrin clot, which could explain how protamine instigates clot lysis and increases bleeding after surgery. Finally, studies in mice reveal that UHRA reverses heparin anticoagulant activity without the lung injury seen with protamine. The data presented here illustrate that UHRA could be safely used as an antidote during adverse therapeutic modulation of hemostasis.

Indoleamine 2,3-Dioxygenase Inhibitors Isolated from the Sponge Xestospongia vansoesti: Structure Elucidation, Analogue Synthesis, and Biological Activity

Two new IDO inhibitory meroterpenoids, xestolactone A (1) and xestosaprol O (2), have been isolated from the sponge Xestospongia vansoesti. Xestolactone A (1) has an unprecedented degraded meroterpenoid carbon skeleton. A short synthesis of the xestosaprol O (2) analogues 3 and 4 features the application of a rarely used photochemical coupling reaction. Synthetic analogue 3 is ∼40 times more potent than the inspirational natural product 2.

Metal ion facilitated dissociation of heme from b-type heme proteins.

Addition of Ni(2+), Cu(2+), or Zn(2+) (10-40 equiv) to metMb in sodium bicarbonate buffer (25 degrees C) at alkaline pH (7.8-9.5) results in a time-dependent (2-6 h) change in the electronic absorption spectrum of the protein that is consistent with dissociation of the heme from the active site and that can be largely reversed by addition of EDTA. Similar treatment of cytochrome b(5), indoleamine 2,3-dioxygenase, and cytochrome P450(cam) (in the presence or absence of camphor) produces a similar spectroscopic response. Elution of metMb treated with Ni(2+) in this manner over an anion exchange column in buffer containing Ni(2+) affords apo-myoglobin without exposure to acidic pH or organic solvents as usually required. Bovine liver catalase, in which the heme groups are remote from the surface of the protein, and horseradish peroxidase, which has four disulfide bonds and just three histidyl residues, exhibit a much smaller spectroscopic response. We propose that formation of carbamino groups by reaction of bicarbonate with protein amino groups promotes both protein solubility and the interaction of the protein with metal ions, thereby avoiding precipitation while destabilizing the interaction of heme with the protein. From these observations, bicarbonate buffers may be of value in the study of nonmembrane proteins of limited solubility.